All viruses tightly regulate cellular processes to create an environment supportive for replication. An understanding of the mechanisms viruses use to do this is vital for our knowledge of viral pathogenesis as well as the normal cellular controls of these pathways. Human cytomegalovirus (HCMV) is a large and slow-growing herpesvirus which must maintain the host cell in a metabolically and translationally active state and circumvent the inhibitory effects of cellular stress responses induced during infection. HCMV induces intracellular reactive oxygen species (ROS) generation soon after infection to facilitate its own gene expression. However, overproduction of ROS can lead to oxidative stress, and it is unclear how the virus controls the detrimental effects of ROS on cell growth pathways. The specific aims of this application are to (1) characterize mitochondrial respiratory chain activity and ROS generation in infected cells throughout a viral time course, (2) examine the effects of oxidative stress on cell growth pathways during infection, and (3) determine the mechanisms that confer resistance to oxidative stress in HCMV infected cells. The Research Design and Methods for this project involve identifying the basal respiration rate and ROS content of cells throughout an HCMV infection. Oxygen consumption is measured using a dark-type oxygen electrode and ROS levels are examined by microscopy and flow cytometry using several different indicators. Next, we will examine the ability of HCMV to resist the effects of oxidative stress. Infected cells are treated with exogenous ROS and cellular signals normally inhibited by ROS will be examined by Western. Finally, pathways important for the maintenance of redox homeostasis, including the antioxidant activities of p53 and Nrf2 and the proteasomal and autophagy pathways, will be examined in HCMV infected cells to identify the mechanisms responsible for protection from oxidative stress. The study of the generation and control of oxidative stress during HCMV infection may facilitate the development of effective therapeutic strategies against this and other viruses. Additionally, these studies may provide valuable insight into key cellular regulatory mechanisms essential for redox homeostasis and their perturbation in disease states.